Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). Cancer is a multistep process, beginning with minor preneoplastic changes, which may under certain conditions progress to neoplasia. Malignant endothelial tumors arise in the setting of autocrine loops involving vascular endothelial growth factor (VEGF) and its major mitogenic receptor vascular endothelial growth factor receptor 2.
Reactive oxygen species (ROS) are believed to be mediators of growth and angiogenesis in cancer. Increased ROS often correlates with cell growth, e.g., Ras-transformed cells and cells treated with growth factors. While non-transformed cells respond to growth factors/cytokines with the regulated production of ROS, tumor cells in culture frequently overproduce H2O2.
NAD(P)H oxidase (Nox) is a cell surface protein with hydroquinone (NADH) oxidase and protein disulfide-thiol interchange activities. In general, most forms of the enzyme can utilize either NADH or NADPH equally efficiently. There are many forms of Nox, including Nox 1-5, Dual oxidase 1 and 2 (Duox 1 and 2), as well as p22(phox), p47(phox) and the small G-protein Rac1.
Nox are believed to account for increased levels of ROS in certain cancers. Reactive oxygen-generating Nox enzymes are implicated in the angiogenic switch, and Nox inhibitors have an effect on ang-2 production in vitro and on bEnd.3 tumor growth in vivo. ang-2 production can be inhibited pharmacologically using Nox enzyme inhibitors, which nearly abolishes bEnd.3 hemangioma growth in vivo. Signal-transduction blockade targeting ang-2 production may therefore be useful for treating human hemangiomas in vivo. Journal of Investigative Dermatology advance online publication, 1 Jun. 2006; doi:10.1038/sj.jid.5700413.
With respect to specific Nox enzymes, it has been shown that transfection of Nox1 into a prostate cancer cell line dramatically enhanced tumor growth (Arbiser et al.: PNAS 99:715-720, 2001), and prostate tumors show increased H2O2 levels. Further, prostate tumors were recently found to show increased levels of Nox1 and hydrogen peroxide (Lim et al., Prostate. 2005 Feb. 1; 62(2):200-7). Nox1-dependent superoxide production has also been shown to control colon adenocarcinoma cell migration (Sadok et al., Biochim. Biophys. Acta. 1783(1):23-33 (January 2008). Sadok showed that Nox1 inhibition or down-regulation led to a decrease of superoxide production and alpha 2 beta 1 integrin membrane availability. Thus, there is a correlation between Nox protein levels and ROS in prostate cancer, and increased Nox1/H2O2 correlates with increased tumorigenicity.
Nox4 is believed to be implicated in inhibition of apoptosis in cancer cells, such as pancreatic cancer cells (Vaquero et al., J Biol. Chem. 2004 Aug. 13; 279(33):34643-54). Vaquero suggested that growth factor-induced ROS produced by NAD(P)H oxidase (probably Nox4) protects pancreatic cancer cells from apoptosis, and that transfection with a Nox4 antisense oligonucleotide inhibited NAD(P)H oxidase activity and ROS production in certain pancreatic cells (i.e., MIA PaCa-2 and PANC-1 cells), and stimulated apoptosis in these cells.
Akt, a signaling molecule downstream of PI3K, is known to induce expression of the ROS-generating enzyme Nox4. One study introduced Akt into a radial growth WM35 melanoma in order to test whether Akt overexpression was sufficient to transform the cells from radial growth to vertical growth. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. Arbiser et al., J. Clini. Invest. 117(10): 2762-2765 (2007). This data supports the premise that inhibition of Akt can inhibit downstream production of Nox 4, which then would inhibit superoxide generation, and therefore treat melanoma.
Duox 1 and 2 are the major Nox species in airway endothelia, and are believed to be one of the main sources for reactive oxygen species production in the airway (Luxen et al., Cancer Res. 2008 Feb. 15; 68(4):1037-45). Accordingly, inhibition of these enzymes may be useful in treating human lung cancer.
Some authors have characterized Nox as falling into two categories. One is hormone-insensitive and drug-responsive (i.e., by quinine-site inhibitors such as capsaicin or the antitumor sulfonylurea, LY181984), designated “tNox,” which is specific to cancer cells. The other is a drug-indifferent constituted form associated with the plasma membrane of non-transformed cells, designated “CNox” (Bruno et al., 1992, Biochem. J. 284:625-628 and Morre and Morre, 1995, Protoplasma 184:188-195).
Cancer cells exhibit both drug-responsive and hormone and growth factor-indifferent (tNox), and drug inhibited and hormone and growth factor dependent (CNox) activities, whereas non-transformed cells exhibit only the drug inhibited hormone- and drug-responsive CNox. Like the tNox of cancer cells, CNox is capable of oxidizing NADH, but has an activity which is modulated by hormones and growth factors. Thus, some authors have theorized that inhibitors of tNox (which are believed to include one or more of the Nox enzymes listed above, such as Nox4) will be useful for treating cancer.
In addition to treating cancer, Nox inhibitors are also expected to have provide therapeutic effects for numerous other inflammatory, degenerative and vascular diseases in which reactive oxygen species have been implicated.
For example, Nox has been reported to have a role in retinal vascular inflammation, as well as ischemia-induced increases in vascular endothelial growth factor (VEGF) and retinal neovascularization (Al-Shabrawey et al., Invest, Ophthalmol, Vis, Sci. (2008)). Studies performed using wild type mice, mice lacking Nox2 and mice treated with the NADPH oxidase inhibitor apocynin in models of endotoxemia and streptozotocin-induced diabetes showed that both endotoxemia- and diabetes-induced increases in ICAM-1 expression and leukostasis were significantly inhibited by deletion of Nox2. Apocynin treatment was as effective as deletion of Nox2 in preventing diabetes-induced increases in ICAM-1, leukostasis, and breakdown of the blood-retinal barrier, suggesting that Nox2 is primarily responsible for these early signs of diabetic retinopathy.
Elevated ROS initiate and anti-oxidants inhibit the apoptotic cell loss in the retinal pigment epithelium (Glotkin et al, 2006 IOVS, 47: 4614-4623). This is thought to play a role in the development of dry age-related macular degeneration. Likewise, the use of antioxidants had been shown to reduce the progression to neovascularization in patients with large drusen in AMD (Coleman and Chew, 2007, Curr. Opin. Ophthalmol. 18(3): 220-223).
NADP+ reductases lower the concentration of retinaldehyde and retinoic acid, which in turn protect cells from retinaldehyde-induced cell death (Lee et al., J. Biol. Chem., 282(49)35621-8 (2007). By extension, inhibition of NADPH oxidase can have the same effect as increasing the rate of a NADP+ reductase, and have a beneficial effect on retinal degeneration mediated by retinaldehyde or retinoic acid.
Specific inhibition of NADPH oxidase has been shown to reduce angiogenesis in models of retinopathy of prematurity (Al-Shabraway et al, 2005, Am. J. Pathol. 167(2): 599-607 and Saito et al, 2007, Mol. Vision, 13: 840-853). In addition elevated ROS have been observed in diabetic animals and the elevation correlates with increase VEGF activity. Similarly, oxidative stress is thought to be a significant factor in the development of diabetic retinopathy (Kowluru and Chan, 2007, Expt. Diabetes Res. Article ID 43603).
ROS may have two separate effects in the development of glaucoma. First, increased ROS led to increased cellularity of the trabecular meshwork (and thereby increased intraocular pressure, Sacca et al, 2007, Exp. Eye Res. 84(3): 389-399). Over time increased reactive oxygen species are also thought to stimulate apoptosis of retinal ganglion cells (Tezel, 2006, Prog. Retin. Eye Res. 25(5): 490-513), the anatomic basis of visual field loss.
In non-ocular cutaneous tissues, NADPH oxidase from pollen has been shown to perpetuate the allergic response Inhibition of NADPH oxidase reduces mast cell degranulation and may be useful in allergic eye disease (Nishikawa et al, 2007, BBRC, 362(2): 504-509).
Although direct experimental evidence that inhibition of NADPH oxidase will provide a therapeutic effect in the some of the eye diseases mentioned is lacking, NADPH oxidase inhibition can be expected to alter the cellular redox balance and thus may be therapeutic in the various condition by indirect means.
NADPH oxidase inhibitors may also be effective for the treatment of dry eye based on the observation that NADPH oxidase is constituitively expressed in corneal epithelial and stromal cells (O'Brien et al, 2006, IOVS, 47: 853-863). The authors suggest that the production of superoxide anion may play a role in inflammation of the cornea.
With respect to the role of specific Nox enzymes in inflammatory disorders, Nox2-containing NADPH oxidase and Akt activation are believed to play a key role in angiotensin II-induced cardiomyocyte hypertrophy (Physiol. Genomics 26: 180-191, 2006).
Accordingly, Nox are believed to be responsible for increased levels of ROS in some cancers and inflammatory disorders, and treatment with appropriate inhibitors may be useful in treating such cancers and inflammatory disorders.
There remains a need for treatment of cancer that does not have the adverse effects generally caused by the non-selectivity of conventional chemotherapeutic agents. There further remains a need to have additional treatments for inflammatory, degenerative and vascular diseases in which a reactive oxygen species has been implicated. The present invention provides such compounds, compositions and methods.